This project proposes to study the mechanism of the initiation of the respiratory burst following phagocytosis by human neutrophils. We shall investigage the role of reduced pyridine nucleotide oxidases in the event. NADH and NADPH oxidase activities will be measured using a fluorometric assay procedure developed in our laboratory. Subcellular localization of the enzyme(s) will be achieved by sucrose gradient centrifugation and by membrane purification. Data to date suggest that the enzyme(s) are localized in a dense granule fraction; this will be further investigated in both normal and CGD cells by isolation of the granule and electron microscopic examination of the fractions. We shall attempt to purify the enzyme activity(s) in order to accomplish two broad objectives: 1) to determine whether two separate oxidases are present or whether a single enzyme is active toward either NADH or NADPH; 2) to permit meaningful kinetic experiments to be performed on the purified enzyme free of possible interfering reactions. Based on data obtained with a very crude neutrophil fraction, we are hypothesizing that NADPH oxidase is an allosteric enzyme and that activation of the enzyme involves an alteration in kinetics from sigmoidal to hyperbolic such that the enzyme can now function at low concentrations of substrate. We shall attempt to prove this hypothesis using purified enzyme prepartions from both normal and CGD cells. Finally, we shall fractionate cells in an attempt to isolate an endogenous mediator of this reaction (i.e., a molecule which might act as an allosteric activator or inhibitor). It is hoped that these approaches will elucidate the normal mechanism of initiation of the respiratory burst and suggest methods which might augment the respiratory burst (and thus the bactericidal activity) of leukocytes in patients with severe infection.